Name: an effective inoculation method for phytophthora capsici on black pepper plants
Uploaded: 2022-09-16T12:40:00
Description: Watch this Scientific Journal Video about An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants at JoVE.com

This work was financially supported by the National Key R&#38;D Program of China (2020YFD1001200), the China Agriculture Research System (CARS-11), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202154), the Natural Science Foundation of Hainan Province of China (321RC652), and the Natural Science Foundation of China (No. 31601626).

1. Preparation of black pepper cutting plants for infection
<ol>
	<li>Take a five-node cutting, approximately 40 cm long with a diameter of 0.5 cm, from a healthy and vigorously growing orthotropic branch of black pepper using a disinfected pruning knife or secateurs. Prune the lower three nodes of the plagiotropic branches, with the upper two nodes left with approximately 10 leaves intact.</li>
	<li>Prepare rooting substrate containing soil and animal manure (cow dung or sheep dung) at a ratio of 1:1....

<ol>
	<li>Gordo, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+black+pepper+root+transcriptome.">High-throughput sequencing of black pepper root transcriptome.</a> BMC Plant Biology. 12 (1), (2012).</li><li>Leonian, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+fruit+blight+of+Peppers+caused+by+Phytophthora+capsici+sp.+Nov.">Stem and fruit blight of Peppers caused by Phytophthora capsici sp. Nov.</a> Phytopathology. 12 (9), 401-408 (1922).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+maize+resistance+by+its+neighbors:+Activating+1,4-benzoxazine-3-ones+synthesis+and+defense+gene+expression+to+alleviate+leaf+disease.">Priming maize resistance by its neighbors: Activating 1,4-benzoxazine-3-ones synthesis and defense gene expression to alleviate leaf disease.</a> Frontiers in Plant Science. 6, 830 (2015).</li><li>Fonseca, C. E. L., Vianda, D. R., Hansen, J. L., Pell, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+among+forage+quality+traits,+vigor,+and+disease+resistance+in+alfalfa.">Associations among forage quality traits, vigor, and disease resistance in alfalfa.</a> Crop Science. 39 (5), 1271-1276 (1999).</li><li>Altier, N. A., Thies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+resistance+to+Pythium+seedling+disease+in+Alfalfa+using+a+culture+plate+method.">Identification of resistance to Pythium seedling disease in Alfalfa using a culture plate method.</a> Plant Disease. 79 (4), 341-345 (1995).</li><li>Pratt, R. G., Rowe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+simplified+leaf+inoculation+procedures+for+identification+of+quantitative+resistance+to+Sclerotinia+trifoliorum+in+Alfalfa+seedling.">Evaluation of simplified leaf inoculation procedures for identification of quantitative resistance to Sclerotinia trifoliorum in Alfalfa seedling.</a> Plant Disease. 82 (10), 1161-1164 (1998).</li><li>Hao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+transcriptome+sequencing+of+black+pepper+(Piper+nigrum+L.)+and+an+analysis+of+genes+involved+in+phenylpropanoid+metabolism+in+response+to+Phytophthora+capsici.">De novo transcriptome sequencing of black pepper (Piper nigrum L.) and an analysis of genes involved in phenylpropanoid metabolism in response to Phytophthora capsici.</a> BMC Genomics. 17 (1), 1-14 (2016).</li><li>Dong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+inoculation+and+classification+of+maize+ear+rot+caused+by+Fusarium+verticillioides.">Field inoculation and classification of maize ear rot caused by Fusarium verticillioides.</a> Bio-protocol. 8 (23), 3099 (2018).</li><li>English, J. T., Laday, M., Bakonyi, J., Schoelz, J. E., &#201;rsek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+molecular+characterization+of+species+hybrids+derived+from+induced+fusion+of+zoospores+of+Phytophthora+capsica+and+Phytophthora+nicotianae.">Phenotypic and molecular characterization of species hybrids derived from induced fusion of zoospores of Phytophthora capsica and Phytophthora nicotianae.</a> Mycological Research. 103 (8), 1003-1008 (1999).</li><li>Chatterjee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activity+of+some+phenolic+constituents+from+green+pepper+(Piper+nigrum+L.)+and+fresh+nutmeg+mace+(Myristica+fragrans).">Antioxidant activity of some phenolic constituents from green pepper (Piper nigrum L.) and fresh nutmeg mace (Myristica fragrans).</a> Food Chemistry. 101 (2), 515-523 (2007).</li><li>Pfender, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+sporangia+and+release+of+zoospores+by+Phytophthora+megasperma+in+soil.">Production of sporangia and release of zoospores by Phytophthora megasperma in soil.</a> Phytopathology. 67 (5), 657-663 (1977).</li><li>Nagila, A., Schutte, B. J., Sanogo, S., Idowu, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chile+pepper+sensitivity+to+mustard+seed+meal+applied+after+crop+emergence.">Chile pepper sensitivity to mustard seed meal applied after crop emergence.</a> HortScience. 56 (2), 1-7 (2021).</li><li>Lamour, K. H., Stam, R., Jupe, J., Huitema, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oomycete+broad-host-range+pathogen+Phytophthora+capsica.">The oomycete broad-host-range pathogen Phytophthora capsica.</a> Molecular Plant Pathology. 13 (4), 329-337 (2012).</li><li>Hardham, A., Gubler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+attachment+of+zoospores+of+a+root+pathogen+and+pre-alignment+of+the+emerging+germ+tube.">Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube.</a> Cell Biology International Reports. 14 (11), 947-956 (1990).</li></ol>

In this study, the basal head was pinpricked to damage and provide an effective inoculation system in the black pepper plant. Mycelial pellets then covered the three holes to retain moisture and enable the pathogen to infect the plant well. Following inoculation, the leaves turned yellow and dropped off and the inoculated plants died. No lesions developed in the control plants. Most genes expressed differently following inoculation with Phytophthora capsici in comparison to the control group. Fungal diseases are...

Black pepper (Piper nigrum L.) is a woody climber and one of the most important spice crops. It is known as the "King of Spices"1 and is cultivated in over 40 countries and regions throughout Asia, Africa, and Latin America. Phytophthora root rot is the most devastating disease of black pepper, and is caused by the oomycete Phytophthora capsici. This pathogen also infects cucurbits, eggplants, chili peppers, and tomatoes2,3. With black pepper, an entire crop can sometimes be decimated by this disease. The expansion of pepper planting areas is restricted as a result of the unavailability of resistant varieties, which has significantly hindered the development of the Chinese black pepper industry. An effective inoculation and a precise sampling system for Phytophthora capsici on black pepper plants are essential for studying this plant-pathogen interaction.
The identification and screening of resistance in germplasm resources is the basic requirement for researching the pathogenicity of the pathogen and the breeding and utilization of resistant varieties. A widely used approach is to use a variety of identification methods based on plant species and pathogen groups. Current identification methods include population identification, individual identification, organ identification, tissue identification, cell identification, biochemical identification, and molecular identification, which have been developed in recent years4,5. There has been success in these areas, but there are also many problems. No matter which method is chosen, the basic requirements of plant resistance identification are consistent, including clear objectives, reliable results, and methods that are simple, rapid, and easy to standardize. This principle must also be followed in the identification of black pepper resistance.
In natural field conditions, the identification of disease resistance can be influenced by many environmental factors. Therefore, it was proposed that detached leaves and irrigated roots be used in the laboratory to identify disease resistance. Young leaves from healthy plants were inoculated in vitro in the laboratory, and the diseased leaves area was measured by inoculating the pathogen in order to identify the disease resistance of plants6. However, in vitro leaf inoculation can only be used for general resistance identification and not for molecular interaction studies. Despite this, the disease-resistant status often presents in the irrigated root inoculation, causing uncertainty in the follow-up study of molecular breeding for disease resistance. Therefore, fast and simple indoor detection methods are essential. This study aims to provide a method for resistance identification in the laboratory.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar powder</td>
			<td>Solarbio</td>
			<td>A8190</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Haier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Xilong Scientific</td>
			<td>15700501</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature sterilizing oven</td>
			<td>Zaelway</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish plates</td>
			<td>Biosharp</td>
			<td>BS-90-D</td>
			<td></td>
		</tr>
	</tbody>
</table>

an effective inoculation method for phytophthora capsici on black pepper plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

Figure 1 shows the symptoms of black pepper leaves following P. capsici inoculation. Figure 2 shows the symptoms of black pepper stems following P. capsici inoculation. The pathogen infected the black pepper at the basal stem; symptoms including leaf yellowing, wilting appearing, xylem browning, and vessel blacking gradually appearing.&#160;Figure 3 shows most of the genes expressed differently after inoculation wi...

Watch this Scientific Journal Video about An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants at JoVE.com

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

The following experiment introduces an effective inoculation and precise sampling method for Phytophthora capsici on black pepper plants. Black pepper, which is a typical woody vine, is an economically important spice crop. Black pepper production is mostly affected by root rot disease caused by Phytophthora capsici, and has seriously influenced the industrial development.An effective inoculation and a precise sampling system for Phytophthora capsici on black pepper plants is important for the study of plant-pathogen interaction. The main goal of this study is to demonstrate a detailed methodology in which the basal head of black pepper was inoculated with Phytophthora capsici, and also provide a reference for the inoculation of woody vine plants. This method provides a better way to solve the instability brought by the traditional inoculation method, such as soil drench or root dipping.Experimental procedures. The cuttings of black peppers are planted in the controlled environment for two months. Subculture the Phytophthora capsici, and incubate for five days under 25 degrees Celsius.On the day of infection, the agar blocks are covered on the three holes sticked by the needle. And finally, characterize disease symptoms. Preparation of black pepper cutting plants for infection.Cut orthotropic branches in 40 centimeter lengths, and 0.5 centimeter of diameter, and removed plagiotropic branches at the lower nodes by sterile knife and secateur. The cut branch should contain two nodes and five leaves at the top to reduce water loss. Trim the upper part to a flat cut and the lower part to an oblique cut.The rooting substrate of black pepper cutting was prepared into a mixture, containing soil and animal manure, such as cow dung, sheep dung, et cetera, at a ratio of one to one. The soil was autoclaved at about 121 degrees Celsius for 20 minutes. Use the breeding bags of 30 centimeter diameter and 50 centimeter height to place the rooting substrates.Prepare a slope of 50 degrees to fill up the substrates. The cuttings were placed at an angle of 50 degrees, with more than three nodes below the substrate. They were covered with the rooting medium and the auxiliary part should be stick to the medium.Finally, the cuttings were fully infiltrated. Straight cutting was adopted to insert the cutting. Preparation of Phytophthora capsici cultures.Choose disease-free potatoes. Clean up the potatoes and cut into small squares. Place 200 grams of potatoes in 800 milliliters of double distilled water, and boiled for 20 minutes.Then filter the mixture. Add 20 grams dextrose. Fill the mixture to one liter with double distilled water.Add 15 grams of agar powder in the filtrate. Dispense the medium into flasks, and seal using Parafilm. Autoclave the medium at 121 degree Celsius for 20 minutes.Calculated medium of potato agar was dispensed into the Petri dish plates, and cooling on the sterile bench for solidification. The inoculation needle was sanitized by heating using alcohol-burning lamp, and cooling for several seconds. Take small pieces of fungus samples using sterile inoculation and place on PDA.The mycelium covers about three quarters of the plate. Cultures are ready for further experiment. Infection of black peppers.The location of five centimeter away from the surface of the substrate was considered as the incubating spot. The incubating location should not be far away from the root, aiming to avoid prolonging the infective time. Detailed steps of pinpricking are as follows.A mycelium pellet of Phytophthora capsici was collected using a stopper borer. Notably, newly developed mycelium pellets on the edge of the plate were preferentially selected because of their higher infective activities. Sterilize a stem using 75%ethanol and take three triangular pores at the sterilized position using the inoculation needle.Three triangular pores at the inoculation location were stuck using the inoculation needle, and mycelial pellets were covered on the three pores. After eight hours, the pores turned black in general. Dilation was enlarged with the prolongation of incubation.After seven to ten days, the leaves turned yellow and drop off, until black pepper were died. Presentative results are as follows. After infection, the symptoms appear on the black pepper plants, including leaf yellowing and wilting, xylem browning, and vessel blacking.Most of the genes expressed differently after inoculation with Phytophthora capsici, compared with the control group. The histopathological analysis of infected tissues has demonstrated Phytophthora capsici colonized in the xylem.Conclusion. In this study, pinpricking the basal head was used to make it damaged to provide a effective inoculation, and the doubling system in black pepper.Meanwhile, this protocol also represents a more efficient way to provide a reference for incubating with-Our method can give more time and operate more briefly. This protocol provides better result, which enables a strong interaction between one plant and the soil-borne pathogen. Which is visible and a convenient to detect the dynamic process between plants and their pathogens.In our method that we begin to pinprick the basal head of the black pepper plant to make it damage them. Then, the damaged area is covered with the Phytophthora capsici directly, and kept the moisture. So that the pathogen could infected the plant.We also provides a promising way for the study of the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

an-effective-inoculation-method-for-phytophthora-capsici-on-black

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...